PROJECT SUMMARY Interaction of the retroviral structural protein Gag with the plasma membrane is a critical step in virus replication. Several principles underlie this interaction, including electrostatic attraction, hydrophobic interaction of a myristoyl group, head group recognition of the signaling lipid PI(4,5)P2 by the MA domain, protein multimerization, interaction with RNA, and possibly recognition of the lipid phase. Also, HIV-1 and Rous sarcoma virus (RSV) Gag also recognize the presence of cholesterol in the membrane. How these principles explain plasma membrane binding and assembly of HIV-1 is incompletely understood. In what will be be the last phase of this long-standing grant, we will address these questions with biochemical, biophysical, and computational techniques, using purified proteins and liposomes of defined composition. We already have purified an arsenal of fluorescent control proteins that act as electrostatic sensors or sensors for certain lipid head groups, as well as various Gag-related proteins. Specific Aim 1 is to systematically test the effects of lipid composition, acyl chain type, and membrane order on interaction of membranes with HIV-1 MA and Gag proteins. These experiments include membrane binding analyses with 100nm liposomes and with giant unilamellar liposomes (GUVs). Experiments also include molecular dynamic simulations (MD) and small angle neutron scattering (SANS). Specific Aim 2 is to use a subset of these purified fluorescent proteins in single molecule analysis on supported bilayers, using total internal reflection microscopy (TIRFM), in order to follow the kinetics of Gag binding and multimerization in real time. Specific Aim 3 is to continue a long- standing collaboration using electron cryo-tomography to obtain higher resolution structures of RSV and equine infectious anemia virus-like particles (VLPs) assembled in vitro, and to explore conditions for reconstitution of a membrane around those VLPs.